Advanced Spectroscopy Technique for Biomedicine - Biomedical Optical Imaging Technologies: Design and Applications - page 19

1.2.6 Spectroscopy System Calibration

A spectrometer must be calibrated before usage. The calibration processes usually
involve two distinct operations: wavelength calibration and spectral response
calibration. For certain quantitative measurements, intensity calibration is also
necessary.

1.2.6.1 Wavelength Calibration

The wavelength calibration of a spectrometer is to correlate the wavelength to the
position of the CCD pixel or the grating position in a monochromator. Wavelength
calibration is usually done with a standard lamp. The most commonly used light
source for wavelength calibration is Hg(Ar) lamp or Hg(Ne) lamp. For Raman
spectroscopy, standard materials, of which the Raman shifts of certain peaks are
already known, can also be used for calibration, such as cyclohexane, acetone, and
barium sulfate. Once the pixel and wavelength pairs are identified, the relationship
can be established by a curve fitting. As the dispersion of the grating is nonuniform,
a fifth-order polynomial fitting is commonly used. A rule of thumb in picking the
pixel-wavelength pairs in wavelength calibration is that the peaks for calibration
should be distributed as uniform as possible to cover the full spectral range.

1.2.6.2 Spectral Response Calibration

Spectral response calibration is more complicated than wavelength calibration. It is
used to correct the transmission properties of the collecting optical components and
the response of the detectors. It is implemented using a NIST traceable standard
lamp, the output of which is assumed to be a blackbody radiator, governed by the
Planck's law. The corrected sample spectrum will be

I./ D I 0 ./ E L ./

I L ./ ;

(1.7)

where E L ./ is the output spectra of the calibration lamp at the specific temperature.
I L ./ is the measured spectra of the calibration lamp using the current spectroscopy
system; I 0 ./ is the measured sample spectrum using the current spectroscopy
system. If the absolute spectral intensity is not important, the spectral correction
factor E L ./=I L ./ may be normalized to its maximum or minimum.

1.2.6.3 Intensity Calibration

In fluorescence and Raman spectroscopy, the spectra are commonly normalized
to its peak intensity or the integrated intensity (area under the curve). These